Identificação molecular de espécies do complexo Anopheles albitarsis (Diptera, Culicidae, Anophelinae), coletadas em dois municípios da Amazônia brasileira, com análise de suscetibilidade natural a plasmódios humanos

Anofelinos membros de complexos de espécies crípticas podem exibir diferenças comportamentais, de susceptibilidade a infecção malárica, e resistência a inseticidas. Assim, a identificação de espécies vetoras tem relevância epidemiológica, o que nem sempre é possível por critérios morfológicos. Métodos alternativos têm sido empregados para tal, como os que analisam regiões altamente conservadas do DNA ribossômico, variável entre as espécies, conhecidas como espaçadoras internas transcritas (ITS). Considera-se atualmente que o complexo Anopheles c seja composto por seis espécies: An. albitarsis s.s., An. oryzalimnetes, An. albitarsis F, An. marajoara, An. deaneorum, e An. janconnae. Destas, pelo menos as três últimas são incriminadas como vetores de malária na Amazônia brasileira. O objetivo deste estudo foi realizar identificação molecular de espécies do complexo An. albitarsis, por análise da seqüências do ITS2 do rDNA, com vistas a analisar sua importância na transmissão de malária nos municípios de Macapá, Amapá e Peixe-Boi, Pará, inclusive investigando pela primeira vez a ocorrência do An. albitarsis F nestas duas áreas epidemiologicamente distintas: a primeira com histórico de alto risco de transmissão de malária e a segunda não. O estudo foi realizado entre janeiro de 2009 e abril de 2010, e consistiu de capturas de anofelinos de 12 horas de duração (ecostofase) no peridomicílio. Todas as fêmeas coletadas foram morfologicamente identificadas e apenas os An. albitarsis s.l. tiveram cabeça e tórax separadas para análise da infecção natural por ELISA; ovários para análise de paridade e patas, asas e carcaça para identificação molecular. Em Macapá foram realizadas seis coletas, obtendo-se um total de 584 anofelinos, sendo 366 An. albitarsis s.l. (62,7%), 167 An. darlingi (28,6%), 33 An. triannulatus s.l (5,6%), 15 An. braziliensis (2,6%) e 3 An. nuneztovari (0,5%). Pela PCR foi possível visualizar a banda específica de An. marajoara em 320 espécimes dos An. albitarsis s.l testados. Do restante, 33 foram negativos e 13 amplificaram um fragmento de ~490 pb nos iniciadores empregados, não permitindo chegar ao diagnóstico específico. O An. marajoara apresentou características biológicas e comportamentais que ratificam sua importância epidemiológica na transmissão de malária em Macapá, tais como: ser a espécie mais prevalente, com maior proporção de fêmeas paridas (73,0%), e portanto com maiores chances de se infectarem com o plasmódio, ocorrer tanto na estação menos quanto na mais chuvosa, e apresentar atividade hematofágica durante toda a ecostofase, alem disso, foi encontrado naturalmente infectado por P. vivax e P. falciparum (taxa de infecção natural de 3,1%). Em Peixe-Boi, foram capturados 43 anofelinos: An. triannulatus s.l (20 espécimes, 46,5 %), An. albitarsis s.l. (13: 30,2 %), An. darlingi (8: 18,6%), e An. nuneztovari (2: 4,7%). Todos os An. albitarsis s.l. coletados foram identificados pela ITS2 como An. oryzalimnetes. Nenhum deles foi encontrado infectado pelos plasmódios testados, e a maioria das fêmeas era parida (84,6%). São necessários levantamentos entomológicos sistemáticos que analisem a importância deste anofelino na transmissão de malária na cidade. O An. albitarsis F não foi encontrado nas duas áreas estudadas. Nossos resultados contribuem para o entendimento da epidemiologia da malária na região Amazônica brasileira.

Culicídeos vetores em uma unidade de conservação da caatinga na região do seridó no Rio Grande do Norte aspectos da transmissão de doenças

The scarcity of occurrence records of culicid species, transmitting insects of etiologic agents for arboviruses, protozoan diseases and philarioses, and lack of information about their interactions with the forest environment make the Caatinga one of the most unknown biomes in Brazil. The aim of this study was to identify culicid species in a conservation unit located in a preserved area of the Caatinga and the possible associations with local abiotic factors, as well as characterize the natural and artificial ecotopes present in the Caatinga of Rio Grande do Norte state. The Ecological Station of Serido (ESEC-Seridó) is a Caatinga Conservation Unit located in a rural area of Rio Grande do Norte. The hot and dry climate of the Caatinga, along with a short rainy season, favor the adaptation and resistance of plants, resulting in completely distinct landscapes over the course of the year. The study took place over the course of one year, with monthly collections between 10h-12h, 14h-16h and 18h-20h. Adult mosquitoes were collected in a forest area using a manual suction trap with human bait as attractant at the three times and a Shannon trap for night collections. During the rainy season ovitraps were randomly distributed at different sites. The different capture sites were observed and photographed. Immature and adult insects captured were taken to the laboratory for identification. Of the 5081 insects collected in human-bait and Shannon traps, 75% were Mansonia wilsoni. In addition, Aedeomyia, Aedes, Anopheles, Coquillettidia, Culex, Haemagogus, Mansonia and Psorophora were identified. Of the 92 Haemagogus eggs found, 71 were collected on the banks of a reservoir. Mosquito larvae were found and photographed in an artificial breeding site of a neighboring rural area. Humidity had the greatest influence on culicid density in the Caatinga. Rain was important in the appearance of Haemagogus, Anopheles, Aedes and Psorophora. Temperature had a negative influence on Anopheles triannulatus. The occurence of important vector species of diseases in a little known preserved area of the Caatinga as well as environmental and sociocultural questions are factors that favor the emergence of transmitting insects, making these areas potential subject to the resurgence of diseases

Two Digestive Trypsins Occur in Three Species of Neotropical Anophelines

Trypsin activity increases in the midgut of Anopheles aquasalis, Anopheles albitarsis, and Anopheles darlingi after a bloodmeal. The activity returns to basal levels at the time the blood is completely digested. Affinity chromatography, reversed-phase high performance liquid chromatography (HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to sequentially purify the mosquito trypsins found in the midguts at 24 h after feeding. Amino-terminal sequencing of the purified trypsins showed the occurrence of two distinct trypsins in the midgut of each of the mosquitoes studied. The sequences obtained are similar to those of the trypsins of other hematophagous insects.

Fauna anofélica da cidade de Belém, Pará, Brasil: dados atuais e retrospectivos

Relato de coletas de anofelinos realizadas em Belém, Pará, Brasil, de 1995 a 2004, comparando os dados obtidos com os de levantamentos anteriores, feitos a partir da década de 1930. Nesses, vinte espécies haviam sido identificadas: Anopheles albitarsis s.l., An. aquasalis, An. argyritarsis, An. braziliensis, An. darlingi, An. eiseni, An. evansae, An. galvaoi, An. intermedius, An. kompi, An. mediopunctatus, An. nimbus, An. nuneztovari, An. oswaldoi, An. peryassui, An. punctimacula, An. shannoni, An. strodei, An. thomasi e An. triannulatus. Sete (An. argyritarsis, An. eiseni, An. galvaoi, An. kompi, An. nimbus, An. punctimacula e An. thomasi) não são agora registradas. A permanência de tantas outras espécies provavelmente decorre da preservação de áreas de mata no âmbito urbano. Duas delas são consideradas de importância vetorial (An. darlingi e An. aquasalis). Esta última continua sendo a de maior densidade nas coletas (46,26% dos adultos e 99,21% das larvas) e é a única registrada em todos os distritos administrativos. Existe, portanto, risco potencial de transmissão de malária em todo o município.

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayestan phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes Within the Albimanus Section the monophyly of the Stroder Subgroup was strongly supported and within the Myzorhynchella Section Anopheles anrunesi and An lutzu formed a strongly supported monophyletic group The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An lanet-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An goeldii were recovered as sister species Finally, there was evidence of complexes in several species, including An antunesi, An deaneorum, and An. strodei (c) 2010 Elsevier B.V. All rights reserved

A importância do Anopheles darlingi Root, 1926 e Anopheles marajoara Galvão e Damasceno, 1942 na transmissão de malária no município de Macapá/AP - Brasil

A malária no município de Macapá é principalmente peri urbana, áreas estas caracterizadas por ressaca, pela presença de fragmentos de floresta e assentamentos desordenados (invasões). O objetivo deste estudo foi verificar a importância dos Anopheles darlingi e Anopheles marajoara na transmissão de malária em Macapá. O estudo foi realizado de outubro de 2007 a setembro de 2008, na comunidade de Lagoa dos Índios, Macapá. Foram coletados 4.601 mosquitos, dos quais 3.029 foram Anopheles marajoara (65,8%), 917 Anopheles darlingi (19,9%), 429 Anopheles braziliensis (9,3%), 208 Anopheles triannulatus (4,5%), 18 Anopheles peryassui (0,4%) e cinco Anopheles nuneztovari (0,1%). Apenas 32,8% dos espécimes foram coletadas no intradomicilio (1.511) e 67,2% no peridomicilio (3.090). O Indice de Picada Homem Hora do An. darlingi no intradomicilio variou entre 0 a 6,5 e no peridomicilio de 0 a 22 picadas homem hora. Já para o An. marajoara a variaçao foi de 0 a 22 no intradomicilio e de 0 a 175,5 no peridomicilio. A analise das exúvias e da dissecção de genitália resultaram na confirmaçao das duas espécies estudadas, An. darlingi e An. marajoara, e que o An. marajoara é a única espécie do complexo albitarsis circulante na área. A abundância dos vetores flutuou associada com o padrão sazonal das chuvas. An. darlingi é mais abundante no final e inicio das chuvas, enquanto o An. marajoara esteve presente em alta densidade durante todo o período de chuvas. Dos 4.601 mosquitos testados, 100 foram positivos para plasmódios humanos pelo método de ELISA, resultando em uma taxa de infecção de 2,17%. Dos 3.029 An. marajoara testados 71 (2,34%) foram positivos e dos 917 An. darlingi, 28 (3,05%). Este estudo demonstrou que as duas espécies estudadas mantêm a transmissão de malária durante todo o ano, ratificando assim a importância das mesmas.

Anopheles gambiae and climate in Brazil

Christopher J Thomas (2003). Anopheles gambiae and climate in Brazil. The Lancet, Infectious Diseases, 3 (6), 326-326. RAE2008

Transglutaminases do mosquito Anopheles gambiae: Caracterização genética, bioquímica e funcional durante a infecção por Plasmodium berghei

A malária constitui um problema de saúde pública, que tem vindo a agravar-se, sendo crescente a necessidade de estratégias renovadas para o seu controlo, como a interrupção do ciclo esporogónico. Deste modo, é essencial compreender as respostas imunológicas de Anopheles anti-Plasmodium. Demonstrou-se anteriormente, que a inibição de transglutaminases, enzimas que participam em vários processos biológicos ao catalisarem a formação de ligações covalentes entre péptidos, agrava a infecção em mosquitos pelo parasita. O presente trabalho tem por objectivo caracterizar as transglutaminases AGAP009098 e AGAP009100 de Anopheles gambiae. Os métodos utilizados para este efeito foram: a sequenciação de regiões dos genes AGAP009098 e AGAP009100; a clonagem molecular de fragmentos da região codificante do gene AGAP009098, usando o vector plasmídico pET–28a(+) e Escherichia coli como sistema de expressão; e PCR em Tempo Real para analisar a expressão relativa dos genes AGAP009098 e AGAP009100 nos diferentes os estádios de desenvolvimento. AGAP009098 é expressa ubiquamente e AGAP009100 a partir do estádio pupa. Estes resultados apontam para a conclusão de que AGAP009098 e AGAP009100 poderão desempenhar funções em processos biológicos relevantes, por exemplo na defesa imunitária, ou no desenvolvimento. Os péptidos recombinantes, obtidos a partir da clonagem com sucesso de fragmentos da região codificante do gene AGAP009098, constituem uma ferramenta importante para averiguar a função destas TGases, no futuro.

Cryptic species status of Anopheles (Diptera: Culicidae) mosquitoes in Canada using a multidisciplinary approach

Many species of Anopheles mosquitoes (Diptera: Culicidae) are now recognized as species complexes whose members are often indistinguishable morphologically but identifiable based on ecological, genetic, or behavioural data. Because the members of species complexes often differ in their vector potential, accurate identification of vector species is essential for successful mosquito control. To investigate the cryptic species status of Anopheles mosquitoes in Canada, specimens were collected from across the country and examined using morphological, molecular, and ecological data. Six of the seven traditionally recognised species from Canada were collected from locations in British Columbia, Quebec, Newfoundland and Labrador, and throughout Ontario, including Anopheles barberi, An. earlei, An. freeborni, An. punctipennis, An. quadrimaculatus s.l., and An. walkeri. Variation in polymorphic traits within An. earlei, An. punctipennis, and An. quadrimaculatus s.l. were quantified and egg morphology examined using scanning electron microscopy. Morphological identification of adult and larval specimens suggested that two described cryptic species, An. perplexens and An. smaragdinus, were present in Canada. DNA sequence data were analysed for evidence of cryptic species using three molecular markers: COl, ITS2, and ITS!. Intraspecific COl variation was very low in most species «1 %), except for An. punctipennis with 2% sequence divergence between those from British Columbia (BC) and Ontario (ON), and An. walkeri with 7% sequence divergence between populations from Manitoulin Island (NO) and Long Point Provincial Park (LP). Similar patterns were also seen using ITS2 and ITS 1. Therefore, molecular data revealed the presence of two putative cryptic species within two species examined (i.e., An. walkeri and An. punctipennis), corresponding to collection location (i.e., NO vs. LP and BC vs. ON, respectively). Surprisingly, there was no molecular support for the presence of either An. perplexens or An. smaragdinus in Canada despite the morphological assessments. Ecological data from all collection sites were recorded and are available in an online database designed to manage all collection and identification data. Current bionomic information, including regional abundance, larval habitat, and species associations, was determined for each species. This multidisciplinary study of Anopheles mosquitoes is the first detailed investigation of these potential disease vectors in Canada and demonstrates the importance of an integrated approach to anopheline systematics that includes molecular data.

Aspects of spatial and habitat ecology of multiple Anopheles species (Diptera: Culicidae): malaria vectors in the highlands and foothills of Ecuador

The resurgence of malaria in highland regions of Africa, Oceania and recently in South America underlines the importance of the study of the ecology of highland mosquito vectors of malaria. Since the incidence of malaria is limited by the distribution of its vectors, the purpose of this PhD thesis was to examine aspects of the ecology of Anopheles mosquitoes in the Andes of Ecuador, South America. A historical literature and archival data review (Chapter 2) indicated that Anopheles pseudopunctipennis transmitted malaria in highland valleys of Ecuador prior to 1950, although it was eliminated through habitat removal and the use of chemical insecticides. Other anopheline species were previously limited to low-altitude regions, except in a few unconfirmed cases. A thorough larval collection effort (n=438 attempted collection sites) in all road-accessible parts of Ecuador except for the lowland Amazon basin was undertaken between 2008 - 2010 (Chapter 3). Larvae were identified morphologically and using molecular techniques (mitochondrial COl gene), and distribution maps indicated that all five species collected (Anopheles albimanus, An. pseudopunctipennis, Anopheles punctimacula, Anopheles oswaldoi s.l. and Anopheles eiseni) were more widespread throughout highland regions than previously recorded during the 1940s, with higher maximum altitudes for all except An. pseudopunctipennis (1541 m, 1930 m, 1906 m, 1233 m and 1873 m, respectively). During larval collections, to characterize species-specific larval habitat, a variety of abiotic and biotic habitat parameters were measured and compared between species-present and species-absent sites using chi-square tests and stepwise binary logistic regression analyses (Chapter 4). An. albimanus was significantly associated with permanent pools with sand substrates and An. pseudopunctipennis with gravel and boulder substrates. Both species were significantly associated with floating cyanobacterial mats and warmer temperatures, which may limit their presence in cooler highland regions. Anopheles punctimacula was collected more often than expected from algae-free, shaded pools with higher-than-average calculated dissolved oxygen. Anopheles oswaldoi s.l., the species occurring on the Amazonian side of the Andes, was associated with permanent, anthropogenic habitats such as roadside ditches and ponds. To address the hypothesis that human land use change is responsible for the emergence of multiple highland Anopheles species by creating larval habitat, common land uses in the western Andes were surveyed for standing water and potential larval habitat suitability (Chapter 5). Rivers and road edges provided large amounts of potentially suitable anopheline habitat in the western Andes, while cattle pasture also created potentially suitable habitat in irrigation canals and watering ponds. Other common land uses surveyed (banana farms, sugarcane plantations, mixed tree plantations, and empty lots) were usually established on steep slopes and had very little standing water present. Using distribution and larval habitat data, a GIS-based larval habitat distribution model for the common western species was constructed in ArcGIS v.l 0 (ESRI 2010) using derived data layers from field measurements and other sources (Chapter 6). The additive model predicted 76.4 - 97.9% of the field-observed collection localities of An. albimanus, An. pseudopunctipennis and An. punctimacula, although it could not accurately distinguish between species-absent and speciespresent sites due to its coarse scale. The model predicted distributional expansion and/or shift of one or more anopheline species into the following highland valleys with climate warming: Mira/Chota, Imbabura province, Tumbaco, Pichincha province, Pallatanga and Sibambe, Chimborazo province, and Yungilla, Azuay province. These valleys may serve as targeted sites of future monitoring to prevent highland epidemics of malaria. The human perceptions of malaria and mosquitoes in relation to land management practices were assessed through an interview-based survey (n=262) in both highlands and lowlands, of male and female land owners and managers of five property types (Chapter 7). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets. In conclusion, this interdisciplinary thesis examines the occurrence of Anopheles species in the lowland transition area and highlands in Ecuador, from a historic, geographic, ecological and sociological perspective.

Larval habitat associations with human land uses, roads, rivers, and land cover for Anopheles albimanus, A. pseudopunctipennis, and A. punctimacula (Diptera: Culicidae) in coastal and highland Ecuador

Larval habitat for three highland Anopheles species: Anopheles albimanus Wiedemann, Anopheles pseudopunctipennis Theobald, and Anopheles punctimacula Dyar and Knab was related to human land uses, rivers, roads, and remotely sensed land cover classifications in the western Ecuadorian Andes. Of the five commonly observed human land uses, cattle pasture (n = 30) provided potentially suitable habitat for A. punctimacula and A. albimanus in less than 14% of sites, and was related in a principal components analysis (PCA) to the presence of macrophyte vegetation, greater surface area, clarity, and algae cover. Empty lots (n = 30) were related in the PCA to incident sunlight and provided potential habitat for A. pseudopunctipennis and A. albimanus in less than 14% of sites. The other land uses surveyed (banana, sugarcane, and mixed tree plantations; n = 28, 21, 25, respectively) provided very little standing water that could potentially be used for larval habitat. River edges and eddies (n = 41) were associated with greater clarity, depth, temperature, and algae cover, which provide potentially suitable habitat for A. albimanus in 58% of sites and A. pseudopunctipennis in 29% of sites. Road-associated water bodies (n = 38) provided potential habitat for A. punctimacula in 44% of sites and A. albimanus in 26% of sites surveyed. Species collection localities were compared to land cover classifications using Geographic Information Systems software. All three mosquito species were associated more often with the category “closed/open broadleaved evergreen and/or semi-deciduous forests” than expected (P ≤ 0.01 in all cases), given such a habitat’s abundance. This study provides evidence that specific human land uses create habitat for potential malaria vectors in highland regions of the Andes.

Aplicación de un sistema de información geográfica para el estudio de la distribución y abundancia de Anopheles Albimanus en el sur de Chiapas, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Control y efectos morfológicos de Anopheles pseudopunctipennis Theobald (Diptera: culicidae) en estanques artificiales del campo agrícola experimental del ITESM, Apodaca, Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Evaluación de dietas y sus efectos en el desarrollo larval de Anopheles pseudopunctipennis Theobald (Diptera: culicidae) en condiciones de laboratorio

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL